Aquaculture becomes more and more important due to the continuous increase of the world population and the exhaustion of the nature fishery day by day. It is estimated that the production of sea fish product in China should reaches 500,000 tons in 2010 so as to meet the demand for sea fish product. It is difficult to sustainably increase production by increasing culture area due to the limited water and soil resources. Therefore, the tendencies of developing sea fish aquaculture in China are large-scale, intensive and high density. However, with stable development of seawater aquaculture, various diseases increasingly arise, resulting in serious effects on the production and development of the culture. In China, the paroxysmal diseases frequently occur in the fish cultivated in net cage or factory in the recent years. Currently, the rate of losses in the aquaculture in China is more than 30%, amounting to an annual loss of over 16 billion RMB. The diseases have become the primary factors that restrict the healthy development of the aquaculture industry.
The chemical therapy using antibiotic has played an active role for controlling, preventing and curing diseases with respect to occurrence of various diseases. However, the negative influences resulting from this kind of control of diseases, such as environment pollution, significant occurrence of drug-resistant pathogens, residual drug in the aquatic products and the like, become more and more serious. The chemical drugs, such as antibiotics as the primary drugs, have been gradually forbidden in the culture fishery in EU, US and CA. According to the stipulations in the White Book of Food Safety in EU, the cultivated aquatic products using antibiotic drugs are forbidden to enter into the trading market in the states of EU. Many antibiotic drugs are also forbidden to use in Japan in the prophylaxis and treatment of diseases in cultivated fish and shrimps.
As a major country in the field of culture fishery in the world, the export of seafood in China only comprises about 6% of the total amount of cultivated products. One of the major confinement factors is that there is serious safety problem in the Chinese aquatic products. The residual amounts of drugs and deleterious agents are seriously above the standard. This is the major obstacle that restricts the expansion of the export. In recent years, the export of aquatic products of China is restricted by EU, Japan and many other nations and regions due to the safety problem. Meanwhile, because the diseases are serious and paroxysmal, in order to reduce the risk of suffering from diseases, the producers generally harvest the product ahead of time, resulting in that product tends to be small. This also affects the rate of export and the export price. To enhance the international market competition of the culture fishery and to enlarge the export of the aquatic products, China has advanced, established and extended the technical standard on nuisance free cultivation, wherein one of the major technical indexes is the prohibition of use of antibiotic drugs, thereby ensuring the safety of aquatic products.
To inhibit the development trend of gradually serious diseases in cultivated fish resultant from various environment factors and the sharp increase of the culture density and the like as well as to improve the sustainable development of the marine culture fishery, the State of World and Agriculture has proposed a so-called System Management Approaches (SMA) culture mode to prevent occurrence of various diseases according to the successful experiences in the fishery development in the developed countries (Ormonde P. Fisheries resources: trends in production, utilization and trade. In: Nomura I (ed.). The State of World Fishery and Agriculture 2002. Rome: FAO Information Division, 2002, p 3-45). One of the major means in this approach is the proposal of use of various vaccination techniques for prophylaxis and treatment represented by vaccine vaccination. Use of these measures will greatly reduce the use of chemical drugs. This will eliminate the environment pollution and improve consume safety of the aquatic products. As an economic and effective disease-controlling policy and means which is in accordance with the environment-friendly and sustainable development strategy, vaccination becomes the major leading and application field in the study and development of the modern aquaculture standard in various countries of the world.
Vaccine exhibits properties of strong pertinence, long period of against disease, absolute immunity, significant effects and positive prophylaxis and treatment. The kill vaccine, which is present in an inactivated form of pathogenic bacteria cell as the basis form, provides effective means for prophylaxis and treatment of diseases of aquaculture. However, the kill vaccine has a common technical application deficiency that its administration is inconvenient. Only by injection administering, can it provide relatively good immunoprotection. This is greatly inconvenient for the fish culture in which thousands of fish need to be vaccinated. The cost of administration is generally not acceptable in the culture fishery. Moreover, it is impossible to administer the fish fry and young fish which are seriously diseased by injection of drugs. Furthermore, the effect of the kill vaccine generally is ineffective or is not good enough to treat many diseases. All of these deficiencies impede the wide use of the prophylaxis and treatment techniques by vaccination in the aquaculture.
According to the industry feature of the culture fishery, it is required that the techniques for the prophylaxis and treatment of diseases should be economic and convenient for operation. Therefore, in addition to the technical requirement of high valence, the development of the vaccine product should have low cost which cannot exceed the capacity for acceptance in the culture industry. The attenuated live vaccine has become the focus and leading field in the current study and development of the aquaculture vaccine in the world due to its new technical advantages, such as convenient administration (it may be administered by immersion), high titer of vaccination (the administration dose may be reduced), low cost, and possibility of developing broad spectrum vaccine (Generally, the live bacterial vaccine possesses cross protection).
Bacteria belonging to the genus Vibrio are the most common pathogens responsible for bacterial diseases in marine culture fishes, such as vibriosis. These bacteria occur at a wide range of temperature, last a long period and cover a wide range of zones. They endanger many kinds of fishes, for example most of the marine fishes such as Percidae, Mugilidae, Bothidae and Pleuronectidae. The pathogens that currently cause relatively serious vibriosis in the cultivated fishes in China mainly include Vibiro anguillarum, Vibiro alginolyticus and Vibrio parahaemolyticus. Vibiro anguillarum mainly and seriously endangers the rare economic fishes, e.g., Bothidae (such as Paralichthys olivaceus and Scophthalmus maximus), Sparidae (such as Pagrosomus major) and Lateolabrax japonicus and the like in the aquaculture fished in northern China, while Vibiro alginolyticus is the main vibriosis pathogen in the primary cultivated economic fishes (such as Epinephelus coioids, and Pseudosciaena crocea etc.) in southern China. These vibriosises are highly dangerous and infectious. There are no effective means for prophylaxis and treatment in China.
Vibrio anguillarum mainly causes hemorrhaging sapraemia in the cultivated fish. The main pathogenic virulence factor of Vibrio anguillarum is the pJM1 like plasmid-encoding iron uptake system in Vibrio anguillarum (Wu H., Ma Y., et al. Complete sequences of virulence plasmid pEIB1 from the marine fish pathogen Vibrio anguillarum strain MVM425 and location of its replication region. Journal of Applied Microbiology, 2004, 97:11021-1028). The inactivated vaccines used for prophylaxis and treatment of vibriosis caused by Vibrio anguillarum have been successfully developed and commercialized. Since the cultivated fish are generally infected by several Vibrio pathogens, the inactivated vaccines available abroad are mainly combined vaccines produced from several vibriosis pathogens. Currently, there are not any types of commercialized vaccines for vibriosis in China which are mainly directed to the prophylaxis and treatment of diseases caused by Vibrio anguillarum. It is reported in U.S. Pat. No. 5,284,653 the utilization of transposon insertion technique for mutation to construct attenuated live Vibrio anguillarum vaccine. There are not any other types of developing attenuated live Vibrio anguillarum vaccine reported in the literature at home and abroad.
The attenuated live vaccine developed by using gene recombination and construction techniques such as transposon and the like contains antibiotic resistant genes and exogenous gene fragments, which may be transferred into the other pathogens. Therefore, they exhibit congenital potential risk of endangering environment. The reversion of virulence and the risk of uncontrollable environment transmission of this kind of vaccines should be taken care and solved when using. In view of these risks, this kind of attenuated vaccine is regarded as biological preparation having relatively high risk of environment safety under the law and regulation on examination and approval executed by the Inspect and Management Institution for Biological Preparation in various countries, including China. Thus, it is difficult to enter into the commercial development procedure.
The technique for constructing the attenuated deletion vaccine without marker gene (or vaccine with unmarked gene deletion) is the newly developing field for development of live vaccine in the world, which is an international leading field and a main development aspect for development of vaccine. The vaccine thus developed exhibits reliable product and environment safety. Moreover, the constructed attenuated strains exhibit potential value of expressing exogenous antigen and developing polyvalent vaccine, especially against diseases caused by virus. Therefore, it becomes an international leading field and a main development aspect for development of vaccine.
The attenuated live vaccine constructed by this technique exhibits the following advantages as compared with those constructed by the traditional techniques:
a) The attenuated live vaccine thus developed does not contain any antibiotic resistant gene. Therefore, it does not exhibit the potential harm resulted from transmission of antibiotic resistance.
b) By using the technique of gene deletion mutation, especially double deletion, it is believed that the virulence cannot revert. As a result, the possibility of transferring a great amount of toxic pathogens to the environment can be greatly eliminated.
c) The genetic background of the mutation is clear, and the attenuation principle is definite. Therefore, it is easy to distinguish the vaccine strain from the wild type strain. As a result, it is convenient to monitor the environment and to improve the environment safety and controllability of vaccine.
All of these technical safety features provide feasible development and application prospect for the commercialized procedure of the attenuated deletion live vaccine without marker gene.